Sensitization of lymphocytes against pooled allogeneic cells. I. Generation of cytotoxicity against autologous human lymphoblastoid cell lines.

Lymphocytes sensitized in vitro to a pool of X-irradiated allogeneic normal lymphocytes from 20 individuals develop cytotoxic activity for autologous human lymphoblastoid cells (LCL). Whereas pool sensitized T lymphocytes lyse autologous LCL cells, they fail to lyse autologous B-enriched or T-enriched normal target cells nor autologous phytohemagglutinin (PHA) blasts. In contrast to pool sensitization, stimulation with normal cells of single allogeneic individuals rarely led to development of cytotoxicity against autologous LCL cells. We conclude that human Epstein-Barr virus transformed LCL cells express target antigens cross-reactive with allogeneic target antigens expressed on normal cells and that sensitization with a pool of allogeneic cells is an effective means of generating effector cells directed against autologous abnormal cells.

Protection of macaques against SIV infection by subunit vaccines of SIV envelope glycoprotein gp160.

Simian immunodeficiency virus (SIV) is a primate lentivirus related to human immunodeficiency viruses and is an etiologic agent for acquired immunodeficiency syndrome (AIDS)-like diseases in macaques. To date, only inactivated whole virus vaccines have been shown to protect macaques against SIV infection. Protective immunity was elicited by recombinant subunit vaccines. Four Macaca fascicularis were immunized with recombinant vaccinia virus expressing SIVmne gp160 and were boosted with gp160 produced in baculovirus-infected cells. All four animals were protected against an intravenous challenge of the homologous virus at one to nine animal-infectious doses. These results indicate that immunization with viral envelope antigens alone is sufficient to elicit protective immunity against a primate immunodeficiency virus. The combination immunization regimen, similar to one now being evaluated in humans as candidate human immunodeficiency virus (HIV)-1 vaccines, appears to be an effective way to elicit such immune responses.

Molecular cloning, sequence analysis, and functional expression of a novel growth regulator, oncostatin M.

Oncostatin M is a polypeptide of Mr approximately 28,000 that acts as a growth regulator for many cultured mammalian cells. We report the cDNA and genomic cloning, sequence analysis, and functional expression in heterologous cells of oncostatin M. cDNA clones were isolated from mRNA of U937 cells that had been induced to differentiate into macrophagelike cells by treatment with phorbol 12-myristate 13-acetate, and a genomic clone was also isolated from human brain DNA. Sequence analysis of these clones established the 1,814-base-pair cDNA sequence as well as exon boundaries. This sequence predicted that oncostatin M is synthesized as a 252-amino-acid polypeptide, with a 25-residue hydrophobic sequence resembling a signal peptide at the N terminus. The predicted oncostatin M amino acid sequence shared no homology with other known proteins, but the sequence of the 3' noncoding region of the cDNA contained an A + T-rich stretch with sequence motifs found in the 3' untranslated regions of many cytokine and lymphokine cDNAs. Oncostatin M mRNA of approximately 2 kilobase pairs was detected in phorbol 12-myristate 13-acetate-treated U937 cells and in activated human T cells. Transfection of cDNA encoding the oncostatin M precursor into COS cells resulted in the secretion of proteins with the structural and functional properties of oncostatin M. The unique amino acid sequence, expression by lymphoid cells, and growth-regulatory activities of oncostatin M suggest that it is a novel cytokine.

A sensitive micromethod for generating and assaying allogeneically induced cytotoxic human lymphocytes.

A sensitive micromethod for generating and assaying allogeneically induced cytotoxic human lymphocytes in vitro is described. Responding lymphocytes are cultured with mitomycin-C treated allogeneic stimulating cells in wells of replicate microtrays for both one-way mixed leukocyte culture (MLC) and cell-mediated lympholysis (CML) assays. On day 5, MLC response is determined by measuring 3H-thymidine (3H-TdR) incorporation directly in wells of the MLC tray. On day 6 or 7 CML response is determined by measuring 51Cr released from labeled target cells added to replicate culture cells in the CML tray. It is thus possible to measure both MLC and CML responses of the 2.5 X 10(4) -U X 10(5) responding lymphocytes originally placed in replicate wells. 51Cr-labeled target cells can be added to wells containing dilutions of the stimulated cells and a log-linear relationship between the per cent specific 51Cr release and number of effector cells is observed. Significant levels of specific cytoxicity are detected at ratios as low as one effector cell per target cell; little cross-killing on third-party cells and no autokilling is observed. Lymphocytes purified from whole blood that is stored overnight at room temperature and purified lymphocytes stored overnight in the cold generate MLC and CML responses comparable to those of lymphocytes purified from fresh blood. Only 2 or 3 ml of whole blood are required to perform both MLC and CML assays, thus enabling the study of both proliferative and cytotoxic lymphocyte responses in young children and other individuals from whom only a few milliliters of blood can be obtained.

Regulation of HIV production by blood mononuclear cells from HIV-infected donors: I. Lack of correlation between HIV-1 production and T cell activation.

The relationship between production of HIV-1 by peripheral blood mononuclear cells (PBMCs) from HIV-1-infected donors and the level of T cell activation by various stimuli was examined. Stimulation of PBMCs with soluble anti-CD3 antibody or staphylococcal enterotoxin/superantigen (SAg) was found to be 100-1000 times more effective at inducing production of HIV-1 than was stimulation with immobilized anti-CD3 or various other T cell activating agents. However, proliferation of CD4+ T cells and lymphokine production following stimulation with soluble anti-CD3 were less than with immobilized anti-CD3. To determine whether immobilized anti-CD3 stimulated cells may produce a factor(s) that suppresses HIV production, dual-chamber coculture experiments were performed in which soluble and immobilized anti-CD3-stimulated CD8-depleted PBMCs were separated by porous membranes. Stimulation of cells by immobilized anti-CD3 suppressed HIV-1 production by soluble anti-CD3-stimulated cells in the inner chamber, suggesting that diffusible factor(s) are involved in suppressing HIV-1 production. Experiments in which exogenous cytokines were added to cells stimulated with soluble anti-CD3 did not reveal the suppressive factor(s) produced; however, IL-7 was found to markedly increase HIV-1 production. Both T cells and monocytes were found to be required for soluble anti-CD3 to induce high levels of HIV-1 production, suggesting a role for adhesion molecules. Our results thus show that (1) soluble anti-CD3 is a powerful stimulus for HIV production, (2) there is not an absolute correlation between the level of HIV-1 production and T cell activation following stimulation of PBMCs with T cell activating agents, (3) immobilized anti-CD3 stimulation produces a factor that decreases HIV replication, and (4) T cell monocyte interactions are important for production of HIV-1 following stimulation with soluble anti-CD3.

Regulation of HIV production by blood mononuclear cells from HIV-infected donors: II. HIV-1 production depends on T cell-monocyte interaction.

Cell-cell interactions induced between T cells and monocytes by certain soluble anti-CD3 monoclonal antibodies (MAbs) were previously shown to be required for high-level production of HIV-1 by peripheral blood mononuclear cells (PBMCs) from infected donors. Staphylococcal enterotoxin or superantigen (SAg) is another mitogen inducing monocytes-T cell interactions that exhibit potent induction of HIV-1 production. Antibodies to several adhesion molecules were used to test the requirements for T cell- and monocyte-associated adhesion molecules in HIV-1 production following activation with anti-CD3 or SAg. Blocking of either CD2-LFA-3, or CD18-ICAM-1, inhibited anti-CD3- or SAg-induced HIV-1 production by more than 90% without inhibiting CD4+ T cell proliferation. Inhibition of HIV production was observed when either the T cell or monocyte coreceptor was bound by MAbs to these adhesion molecules. Blocking of CD28-B7 interactions by soluble CTLA-4 fusion protein, a CD28 homolog, inhibited both HIV-1 production and CD4+ T cell proliferation. Fc binding was not required for HIV-1 inhibition by MAbs to CD2 and CD18, because Fab or F(ab')2 fragments of these MAbs inhibited HIV-1 production by more than 80%. A chimeric single-chain MAb to CD2 was produced, containing heavy and light chain variable regions from MAb 35.1 to CD2 linked to the constant regions of human IgG1 (CD2 SFv-Ig). This humanized CD2 SFv-Ig inhibited HIV-1 production by 30% to > 98%. These results thus indicate that simultaneous engagement of multiple adhesion pathways between T cells and monocytes are required for HIV production by patients PBMCs and may have implications for therapy of HIV infections.

Hepatitis B surface antigen-specific CD8-positive T cell clones. Antigen specificity and interleukin-2 production.

Clones of hepatitis B surface antigen-reactive CD8+ and CD4+ T cells were obtained from peripheral blood mononuclear cells (PBM) of a hepatitis B immunized individual whose PBM proliferated when cultured with hepatitis B surface antigen (HBsAg). Lymphocytes were activated by culturing for 2 weeks with HBsAg and high concentrations of interleukin-2 (IL-2), then cloned in the presence of irradiated HBsAg-activated PBM and autologous Epstein-Barr virus (EBV)-transformed B cells, together with antigen and IL-2. All clones examined proliferated in an antigen-specific manner. Of 7 clones examined by flow cytometry, 4 were CD4+, CD8-; and 3 were CD4-, CD8+. Several clones produced IL-2 activity after stimulation with hepatitis B surface antigen. Since development of CD8+ T-cell clones specific for soluble antigens is difficult, the high frequency with which CD8+ cells were cloned in these experiments suggests that the cloning strategy employed might have general use for development of CD8+ clones. Availability of hepatitis B virus specific T cell clones of different phenotypes may help elucidate mechanisms of immunotolerance in hepatitis B infection.

Impaired natural killer activity in patients with chronic lymphocytic leukemia is associated with a deficiency of azurophilic cytoplasmic granules in putative NK cells.

This study was undertaken to gain further insight into the severely impaired natural killer (NK) activity we and others have previously observed in patients with chronic lymphocytic leukemia (CLL). Normal individuals' NK cells are large granular lymphocytes (LGL) that (A) bind to and lyse NK-sensitive cells, including K562, (B) express receptors for the Fc portion of IgG (FcR+ cells), and (C) express cell surface antigens reactive with monoclonal antibodies OKM1, 9.6, and OKT11A. We thus examined lymphocytes depleted of monocytes and B cells, from 6 CLL patients and 6 normal individuals, that were identified on the basis of binding to K562, expressing OKM1, or expressing receptors for the Fc portion of IgG. In the CLL patients studied, lymphocytes that bind to K562 cells, as well as OKM1+ cells isolated by fluorescence activated cell sorting, were morphologically similar to LGL of normal individuals, with the exception that more than 75% of the patients' cells were deficient in azurophilic cytoplasmic granules, which typify normal individuals' LGL. Furthermore, although the percentages of the patients' FcR+ cells reactive with OKT11A, 9.6 and OKM1 were very similar to those of normals, the majority of the patients' FcR+ cells were deficient in azurophilic granules and lacked NK activity. These findings indicate that the impaired NK activity in CLL patients is associated with cells that are phenotypically and morphologically NK cells, but which lack azurophilic granules that are thought to play a role in NK-mediated lysis.

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. I. Lysis restricted by HLA class II MB and DR antigens.

In contrast to general findings that mouse and human cytotoxic T lymphocytes (CTL) are restricted in cytotoxic activity by major histocompatibility complex (MHC) class I antigens, we previously found that some herpes simplex virus (HSV) type I-infected cells that shared no HLA class I antigens with the HSV-1-stimulated lymphocytes were lysed. In this study, we addressed the question of the role of HLA antigens in human T cell-mediated lysis of HSV-1-infected cells by generating clones of HSV-1-directed CTL from two HSV-1-seropositive individuals. CTL clones that lysed autologous HSV-1-infected lymphoblastoid cell lines (LCL), but not natural killer-sensitive K562 cells or uninfected or influenza virus-infected LCL, were tested for cytotoxicity against a panel of allogeneic HSV-1-infected LCL. Clone KL-35 from individual KL lysed only HSV-1-infected LCL sharing the HLA class II MB1 antigen with KL. With all four CTL clones isolated from individual PM, only HSV-1-infected LCL sharing DR1 with PM were lysed. Monoclonal antibody s3/4 (directed against MB1 ), but not TS1/16 or B33 .1 (directed against a DR framework determinant), blocked lysis of autologous HSV-1-infected cells by KL-35. In contrast, B33 .1, but not s3/4, blocked lysis of autologous HSV-1-infected cells by the PM CTL clones but not by KL-35. Together, these results indicate that our five human CTL clones which are directed against HSV-1-infected cells, and which are all OKT3+, OKT4+, OKT8-, are restricted in lytic activity by HLA class II MB and DR antigens. These results suggest that the HLA D region-encoded class II antigens may be important in the recognition and destruction of virus-infected cells by human CTL.

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. III. Analysis of viral glycoproteins recognized by CTL clones by using recombinant herpes simplex viruses.

Human cytotoxic T cell (CTL) clones specific for herpes simplex virus (HSV) type 1- and type 2-infected cells were generated and were analyzed with regard to the viral glycoproteins they recognize on autologous HSV-infected cells. By use of target cells infected with wild-type HSV strains, a gC deletion mutant of HSV-1, and HSV-1 X HSV-2 intertypic recombinants, some HSV-1-specific CTL clones were found to be directed against L region-encoded gA/B-1, and others against S region-encoded glycoproteins (gD-1 or gE-1). Some HSV-2-specific clones were found to be directed against L region-encoded gC-2, whereas others were directed against S region-encoded glycoproteins (gD-2, gE-2, or gG). These findings provide direct evidence that several HSV glycoproteins that are expressed on the surface of HSV-infected cells serve as recognition structures for human HSV-specific CTL.

Alloantigen-induced enhancement and suppression of human cytotoxic lymphocyte responses to autologous lymphoblastoid cell lines.

In vitro sensitization of human lymphocytes to autologous lymphoblastoid cell lines (LCL) has been shown to give rise to cytotoxic lymphocytes capable of lysing autologous as well as allogeneic LCL cells. However, allogeneic LCL cells were found to be markedly less effective than autologous LCL cells in terms of generating lymphocytes capable of lysing autologous LCL cells. The addition of allogeneic LCL cells or allogeneic normal lymphocytes to a mixture of responding lymphocytes and X-irradiated autologous LCL cells suppressed the generation of cytotoxic lymphocytes against autologous LCL cells. Furthermore, suppressor T cells generated in allogeneic mixed leucocyte culture (MLC) and supernatants from MLC likewise decreased the generation of cytotoxic lymphocytes to X-irradiated autologous LCL cells. In contrast to the findings that alloantigens suppress the generation of cytotoxicity of X-irradiated autologous LCL cells, which ordinarily induce strong cytotoxic responses, were the findings that allogeneic stimulating cells and supernatants from MLC enhanced cytotoxic responses to autologous ultraviolet light or extensively heat-treated LCL cells that induce weaker cytotoxic responses. The possible mechanisms whereby alloantigens enhance or suppress cytotoxic responses to autologous abnormal cells and the implications of these findings are discussed.

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures.

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.